Beta-lactamases are enzymes produced by some bacteria. They are responsible for bacterial resistance towards beta-lactam antibiotics (e.g. penicillins, cephalosporins, carbapenems). A suitable method for the determination of bacterial resistance is detection of beta-lactamases. It is important for an effective antibiotic treatment and for setting of suitable precautions to block the spreading of their producers.
Beta-lactamase identification is carried out using phenotype methods based on the sensitivity towards different inhibitors. A precise identification takes place by the use of PCR amplification of the genes thereof followed by sequencing of the amplicons. There are several laboratories dealing with the development of beta-lactamase identification methods using mass spectrometry (e.g. Schaumann R, Knoop N, Genzel G H, Losensky K, Rosenkranz C, Stingu C S, Schellenberger W, Rodloff A C, Eschrich K. 2012. A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry. Med. Sci. Monit. 18: MT71-MT77).
The method of protein (beta-lactamase) detection using MALDI-TOF (matrix assisted laser desorption/ionization time-of-flight) mass spectrometry is capable of affording results comparable with molecular genetics assays (PCR, microchips), yet making the whole process considerably faster and cheaper. So far, no effective method of detection of these enzymes using MALDI-TOF mass spectrometry has been published. The only published work, in which beta-lactamase has been identified using MALDI-TOF mass spectrometry, is the work of Camara J E, Hays F A. 2007. Discrimination between wild-type and ampicillin-resistant Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Bioanal. Chem. 389: 1633-1638 from 2007. The results published by the above-mentioned authors were, however, irreproducible. At the same time there exist no publications confirming the conclusions of the above-cited work.
It is known from WO 2012/143535 and WO 2012/143534 that carbapenemases and cephalosporinases can be detected by mass spectrometry, however, the proteins are first cleaved into short peptides and the type of carbapenemases and cephalosporinases is then determined according to the spectra typical for those peptides.
Furthermore, the detection of bacterial resistance is known, wherein products of treatment with enzymes causing the resistance are followed by mass spectrometry. It involves covalently modified antibiotics or covalently modified model compounds (US 2012/196309). Likewise, there is known a determination of the presence of beta-lactamases, based on the detection of products of beta-lactamase activity, thus based on the detection of products of hydrolytic cleavage of the beta-lactam ring amide bond of beta-lactam anibiotics (WO 2011/154517).
All methods of detecting the enzymes of bacterial resistance known heretofore require either enzyme degradation into peptides or carrying out of the enzymatic reaction followed by the detection of the products of this reaction. This generates the risk of false positive results by incorporating additional components into the mixture being analyzed.